DE3885021T2 - Automatic measurement method for glycohemoglobin. - Google Patents

Description

The present invention relates to an improved automatic measuring method for glycated hemoglobin based on the principle of high pressure liquid chromatography

Glycohemoglobin (HbA1) for fixing glucose to hemoglobin is often found in diabetes patients. In particular, HbA1c is an important measurement substance as an index for a health check-up such as a medical check-up or for long-term diabetes monitoring, because HbA1c is abundant in glycohemoglobin (HbA1) and the increase of this substance due to diabetes is much larger than that of the other substance. In addition, the value of HbA1c has a significant correlation with the average fasting blood sugar level over the past 1-3 months.

Glycohemoglobin includes HbA1a and HbA1b in addition to HbA1c. These components are measured by colorimetry, electrophoresis, mini-column techniques or high-pressure liquid chromatography. Of these techniques, high-pressure liquid chromatography (HPLC) has recently become the dominant method in clinical testing due to the time and separation required.

A so-called stable type of HbA1c has a significant correlation with a past blood sugar level, which is not the case with a labile type. The proportion of the labile type is about 10-15% of the total HbA1c in the hungry state of a healthy person. In this labile HbA1c, the N-end of the β-chain of hemoglobin and the reductive end of glucose form a reversible Schiff base combination, which forms and decomposes in a relatively short time depending on the blood sugar level. Therefore, it is present in diabetes patients more than in healthy patients and sometimes amounts to 10-20% of the total HbA1c. After a meal, its proportion is greater than in the hungry state, which is greatly influenced by the state at which the blood is taken.

Stable HbA1c is gradually, continuously and irreversibly generated from labile HbA1c, reflecting long-term blood glucose levels in the past. Thus, only the separate measurement of the stable type is desirable. However, the stable and labile types are structurally very close, making it quite difficult to separate them by liquid chromatography.

To overcome these difficulties, separation was carried out using a long high-resolution column. Although this method has the advantage that denaturation of glycated hemoglobin by chemical treatment cannot occur, an analysis time of 10 or several minutes or more is required to obtain a good separation, so that an increasing number of samples or an urgent measurement cannot be handled adequately. In addition, this type of column is very long, resulting in a large-scale device and high costs.

Another method for measuring stable HbA1c separately involves removing labile HbA1c by chemical decomposition in a pretreatment. This is based on the fact that the temporary combination (Schiff base) of labile HbA1c and glucose can easily decompose. For example, washed red cells are incubated in isotonic phosphoric acid buffer solution (37ºC, 4 h) or in physiological saline solution (room temperature, 14 h) to release glucose from labile HbA1c.

There is also a method in which whole blood added to a hemolyzing reagent is incubated at 35ºC for 10 or several hours. This reduces the proportion of labile HbA1c by hemolyzing and diluting a sample, which has a particularly large effect in the acidic range below pH 6 with a fast reaction rate and a large effect at elevated temperature. By adding a commercially available reagent containing boric acid to remove labile HbA1c, which was used for the mini-column method, this effect is increased.

However, this pretreatment requires time, and together with the decomposition of the labile HbA1c, the decomposition and modification of the other glycated hemoglobin and of pure hemoglobin (HbA�0) takes place at the same time.

Such a hemolysis method has the defect that the amount of stable HbA1c varies with the time elapsed after hemolysis and the temperature that occurred before the measurement. In particular, in the case of automatic measurement of a series of samples, there is the disadvantage that the dilution of the blood sample by the hemolysis fluid, which contains a decomposing agent, the sample measured later changes from the available samples due to excessively rapid progression of the reaction because of the longer time that has elapsed.

An automatic analysis method for measuring hemoglobin using high-pressure liquid chromatography is known from Clinical Chemistry 25, 1970-71 (1979). This method corresponds to the state of the art described above.

From Clinical Chemistry 27, 1261-63 (1981) it is known that the unstable hemoglobin fraction can be removed by either washing it out or incubating it at 250 for 14 hours with an isotonic saline solution. However, this results in an undesirable and uncontrolled decomposition of the stable glycated hemoglobin fractions that are to be measured.

The object of the present invention is to provide a method for measuring stable HbA1c with good reproducibility and high accuracy, in which the decomposition and change of glycated hemoglobin other than the labile HbA1c is prevented, together with the analysis of glycated hemoglobin using an agent (decomposing agent) for removing the labile HbA1c by high pressure liquid chromatography. Furthermore, the invention is to provide a method for analyzing glycated hemoglobin in which the required device has a relatively simple structure and the treatment and preparation of the sample are simple.

In addition, the invention aims to provide an optimal sample injection valve for controlling the temperature of the mixed solution the sample (not limited to a blood sample) and the reagent in a high pressure liquid chromatograph.

This object is achieved by a method according to patent claim 1.

Improvements to this method emerge from the subclaims.

From the drawings show:

Fig. 1 is a diagram showing an embodiment of an automatic measuring device for glycated hemoglobin for carrying out the method according to the invention;

Fig. 2 is a diagram showing the sampling part;

Fig. 3 a spatial representation of the sample injection valve; and

Fig. 4 is a plan view of the sample injection valve in a state where the sample is injected into the column.

According to this, the term "sample" refers to the entire blood sample (or blood cell layer) and the term "specimen" refers to the mixed fluid consisting of the diluted sample and the hemolysis fluid.

The reaction is accelerated by heating until the mixed liquid (specimen) is injected into the column, allowing for faster measurement. If this heating is performed on the loop part of the sample injection valve, there is no need to create another heating zone or unnecessary heating of the specimen, thus saving space and energy.

According to the invention, time for manual mixing of the reagents is also saved and errors in manual mixing and changes in the sample until measurement after mixing are avoided.

The method of the invention and the structure of the measuring device are described below.

First, blood samples collected from each patient or subject are placed in sample containers such as blood tubes or sample cups and left standing on the sample holding part as blood or as a blood cell layer. Since whole blood or the blood cell layer centrifuged from whole blood is used, pretreatment is not required and is simple. The blood cell layer can be used by making use of the blood plasma remaining after the other tests. However, whole blood also deposits blood cell components at the bottom after standing for a long period of time. Therefore, sampling is preferably carried out from near the bottom. Although changes occur in the proportion of red cells in the blood sample, the ratio of HbA1c to HbA0 or the other HbA1 is constant in each sample, so this does not pose a problem. An anticoagulant is usually added to the blood sample. Commercially available agents, such as heparin or EDTA-2 Na, can be used as anticoagulants.

Each sample vessel is held in a holding device, such as a rack, a queue or a turntable, in a large number and guided one after the other to the sampling position.

At the sampling part, a specific amount (1 to a few μl) of the sample is sucked out through a sampling nozzle by the suction effect of a pump and diluted and mixed with hemolyzing fluid containing a separately added reagent for removing labile HbA1c. The dilution is 10-100 times, preferably 10-400 times.

As for the sampling and dilution methods, various designs and changes are possible depending on the designs and combinations of various types of pumps. In short, a design is preferred in which the blood samples taken within several tens of seconds to several minutes immediately before measurement are diluted with a hemolyzing washing liquid and injected into the column through the sample injection valve at a specific time after the start of mixing. It is necessary that these operations be carried out automatically and continuously, and a means is proposed that can avoid contamination.

Boric acid, phosphorus compounds or commercially available reagents containing these substances are used as reagents for removing labile HbA1c. Substances with an acidic pH value are preferred. Commercially available agents are used as hemolyzing agents.

The column used is one for high-pressure liquid chromatography, which is filled with a spherical ion exchange gel (anion, cation). The treatment time of the specimen is limited by the separation.

The reaction state after hemolyzing depends on the properties of the agent for removing labile HbA1c, and the time and temperature after the start of the reaction. The time for measurement is the sum of the operation time for sampling, dilution, mixing, feeding and washing, and the heating time. A short measurement time is preferred to treat a series of samples. When the ability of the reagent to decompose labile HbA1c is low, shortening the reaction time is carried out by heating the specimen flow (mixed liquid) to a temperature range as in the sample change. In this case, the temperature of the entire flow or a part of it, including the sample injection valve part, is controlled so that the time is kept constant with respect to the analysis time and the decomposition ability of the reagent until measurement. In view of the decomposition capacity and the heating time, the heating temperature is 30-65ºC, preferably 40-55ºC. A temperature above 65ºC causes an undesirable change in the protein. A temperature below 30ºC may require refrigeration in summer.

The washing liquid is described below. The washing liquid is used to wash the sampling nozzle and each run to prevent contamination of the last specimen after measurement. The design of the device according to the invention, in which the sample is mixed with the hemolyzing liquid after dilution, naturally requires two supply systems with respect to the washing liquid. and the hemolyzing fluid. It goes without saying that such a design can be used. However, both fluids can also be combined as a hemolyzing wash fluid, thereby eliminating one of the feed pumps and simplifying the tubing and feed sequence.

According to the invention, the mixed liquid (specimen) is injected into the column at a specific time after the start of mixing of the blood sample and the hemolyzing liquid. In the case of starting mixing at the wrong time when counting back from the next injection time for some reason such as an unsmooth shift of the rack or an interposed measurement at the wrong time, a blank measurement without mixing and injection at that time is preferred. Such mixing at the wrong time does not cause any problems if only one type of eluting solution is used. However, if two or more types of eluting solution with different concentrations or different pH values are used, the injection at the wrong time prevents equilibration in the column.

Example

The present invention is described in more detail below in connection with the following example of a measuring device for glycated hemoglobin for carrying out the method of the invention. In this example, a hemolyzing liquid is used, which also serves as a washing liquid, and a specimen is heated in the sample loop at the sample injection valve.

The device consists of a sample holding part 1 for holding and providing a plurality of sample vessels containing a whole blood sample or a blood cell layer, a sample taking part 2 for taking the samples and diluting/mixing them with the hemolyzing liquid, a bottle unit part 3 for accommodating vessels for an eluting solution, hemolyzing/washing liquid and waste solution, an analyzing part 4 including a sample injection valve, a column and photometric devices, a memory control part for controlling the operation of the entire device, storing and calculating measurement values and outputting the results to a display device such as a printer together with a patient number and measurement data, and a keyboard for inputting operation commands. A microcomputer 5 is used as the memory control part, for example. A digital display device can be used for the display device.

The structure and operation of each part is described below. In the sample holding part 1 there are a series of racks 7 which hold a plurality of sample vessels 6. Each rack 7 is driven in the direction of the arrow so that the sample vessels 6 reach the sample taking position one after the other. A measuring opening 8 for urgent measurements can be provided.

The sampling part 2 has a sampling nozzle mechanism 11 with two nozzles 9, 10, a hemolyzing/washing liquid pump P₁, a sample suction pump P₂, a specimen injection pump P₃ and a pouring vessel 12 for dilution. The capacity of the sample suction pump P₂ is 1 to several µl, and that of the washing liquid pump P₁ and the specimen injection pump P₃ is hundreds of pl. The sampling nozzle mechanism 11 moves up and down with one rotation. The Hemolyzer/washing liquid 13 is prepared by dissolving the above-mentioned decomposer for labile HbA1c and hemolyzer in the diluted sample liquid. In the embodiment, this liquid is also used for washing the pumps, nozzles and other flow lines. In the invention, the term "sample" 14 refers to a whole blood sample or a blood cell layer, while the term "specimen" 15 refers to a solution prepared by diluting the sample 14 with the washing liquid 13 up to 10-100 times.

At the sampling part 2, first, a specific amount of a sample 14 is sucked from the first nozzle 9 by driving the sample suction pump P2. Next, the sampling nozzle mechanism 11 is moved over the pouring vessel 12 for dilution, and the washing liquid (13) is discharged from the second nozzle (10) to wash the outside of the first nozzle 9. For this purpose, the second nozzle 10 has a tip bent toward the first nozzle 9. The waste liquid is supplied through a waste liquid valve 16 to a drainage bottle 17 at the bottle unit part 3. Next, the sample 14 and a specific amount of the hemolyzing/washing liquid 13 are discharged into the pouring vessel 12 for dilution by driving the pumps P1 and P2. Both the liquid 13 and the sample are thereby stirred. Both can be sufficiently stirred and mixed by repeated suction and discharge through the first nozzle 9. Thus, a mixed specimen 15 is sucked from the first nozzle 9 by the suction of the specimen injection pump P3 and is introduced into the sample injection valve 18 on the analyzing part 4 (state of Figs. 1 and 2). Next, the pouring vessel 12 for dilution, the tubes and the outside the first nozzle 9 with the hemolyzing/washing liquid 13 and the waste liquid is discarded. Preparations are made for aspirating the next sample.

The specimen 15 introduced into the sample loop 18a at the sample injection valve 18 is heated by being held in the heated sample loop 18a. As shown in Fig. 3, the sample loop 18a is surrounded by a loop heater 18b. The loop heater 18b and the main part of the sample injection valve 18 are covered by an insulation cover (temperature holding cover) 18c. In the figure, 18d is a tube for the specimen, 18e is a tube for the eluting solution, 18f is a motor, 18g is a gear, and 18h is a block for holding the temperature. The insulation cover 18c is screwed to the block 18h.

When heating using this method, no unnecessary energy is required and no unheated specimen part is injected into the column 19, since safe heating of the specimen only takes place in the sample loop 18a.

Next, the motor 18f of the sample injection valve 18 is rotated to the state shown in Fig. 4, and the specimen 15 in the sample loop 18a is injected into the column 19 by being expressed by the eluting solution supplied from the bottle unit part 3. Each component of the specimen 15, HbA1a, HbA1b, HbA1c and HbA₀, is separated in the column 19 and subjected to photometry one by one by a photometer 20 and discharged into the waste vessel 21. The photometric results are supplied to the microcomputer 5, and the pattern of each fraction which Peak elution time as well as the percentage of each component are calculated to be printed out by a printer 22. When the filter 24 is placed in front of the column 19 to remove impurities and the whole is kept in a thermostat 25, stable measurements are made.

Table 1 shows the summary of the above-mentioned processes on the basis of Fig. 2 concerning the operation of the pumps P1, P2 and P3 and the state of the valve 38, 39 for the hemolyzing/washing liquid 13. For each pump, ↓ means suction, ↑ delivery and stop. Table 1 Pump, Valve Step Operation Sample is aspirated, hemolyzer/wash liquid is aspirated Hemolyzer/wash liquid is dispensed to wash the nozzle outside Hemolyzer/wash liquid is aspirated Blood sample and hemolyzer/wash liquid are dispensed for mixing Specimen is aspirated and sent to the sample loop for heating Specimen is injected into the column by replacing the sample injection valve, remaining specimen is dispensed Sample loop and other flow lines are washed

In the above-mentioned mode of operation, the 450 µm hemolysing/washing fluid 13 for diluting 1.5 µl of the sample 14 has a 300-fold dilution. The optimum conditions of heating time and temperature of the specimen 15 are 2 min 40 sec at 48ºC, although they depend on the decomposition ability of the reagent for removing the labile HbA1c. In the above-described dilution, a hemolysing fluid containing a reagent for removing the labile HbA1c from a phosphoric acid compound, sold by the patentees under the designation "21H", was used. The decomposition of the labile HbA1c was almost completely achieved at 2 min and 60ºC, 3 min and 40ºC and 4 min and 33ºC.

In the embodiment, three kinds of eluting solution are used. Each eluting solution A, B and C is supplied according to the flow sequence to a distributor (34) arranged after each exchange valve 31, 32 and 33 through a heating coil 26, cooling coil 27 and bubble removing device 28, 29 and 30. Each eluting solution entering a flow line on the distributor 34 is supplied to the sample injection valve 18 via the supply pump 35, injected into the column 19 and flows into the dehydration vessel 21 through the photometer 20 carrying the specimen 15. In the figure, 36 denotes a pressure detector and 37 denotes a slide valve.

In Fig. 1 and 2, 38 and 39 designate an exchange valve for the hemolyzing/washing liquid 13, 40 a sensor for the hemolyzing/washing liquid 13 to indicate that no liquid is present, 41 an intake air pump for the waste water bottle 17, 42 a display and 43 a keyboard.

The above description refers to the measurement of samples that are arranged continuously. The resulting operation in a constant sequence leads to a constant time from dilution to injection. However, if vessels are not arranged continuously on the rack 7 because of different times with respect to the withdrawal of blood, an irregular amount of time is required to find the next sample. Also, in the case where a sample vessel is inserted into a corresponding opening for an urgent measurement, erroneous aspiration of the blood sample may occur. Thus, when the injection of the specimen 15 into the column 19 is completed, the next sample is determined and the dilution is preferably started at the right time by counting back from the next injection time.

At the start of dilution, when the absence of the next injection is already clear, the continuous operation of a sequence without mixing and injecting the specimen into the column achieves the continuation of the continuous measurement without affecting the column equilibrium state, even in the case where three types of eluting solutions of different concentration or pH are used.

Comparison with the conventional method

(Comparison with the blood cell washing procedure using an isotonic phosphoric acid buffer solution)

The blood cell washing procedure over an isotonic phosphoric acid buffer solution, which was used as a standard procedure for removing labile HbA1c (pretreatment), was compared with the method of the present invention. Table 2 shows the corresponding results. Table 2 Proportion Condition Measured value F: Glycohemoglobin HbF LA₁ c: labile HbA₁ c SA₁ c: stable HbA₁ c "S": Blood cell washing procedure with isotonic phosphoric acid buffer solution

It can be seen from the table that the method according to the invention can achieve effects similar to those of the conventional method in terms of removing the proportion of labile HbA1c. Furthermore, in comparison with the combination of the blood cell washing method and 21H, no greater reduction in the proportion of labile HbA1c and no increase in Ala+b were observed.

In the case of no pretreatment and no use of reagent 21L to remove labile HbA1c, all values were high. The difference between 21L and the other treatments in L-A1c+S-A1c is considered to be the labile HbA1c. In the example, labile HbA1c accounts for about 0.7% of total hemoglobin. Measurements were performed under the following conditions:

21L: Hemolyzer/washing fluid without reagent for removing labile HbA1c

Non-ionic surfactant Ig/l

Potassium dihydrogen phosphate 0.1 g/l

Potassium monohydrogen phosphate 0.3 g/l

pH: 7.5.

21H: Hemolysis wash fluid containing a reagent to remove labile HbA1c

Non-ionic surfactant: 1 g/l

Phosphoric acid compound: 0.1 g/l

KOH: 0.3 g/l

pH: 6

"S" + 21L: Conventional blood cell washing procedure Washed blood cells were added to isotonic phosphoric buffer acid and labile HbA1c was determined by incubation over 6 h at 37º with continuous rotary mixing.

Then the blood cell layer was collected and diluted 300-fold with 21L.

"S" + 21H: 21H in combination with the blood cell washing procedure

In a similar manner to "S" + 21L, the blood cell layer treated with isotonic phosphoric acid buffer solution was collected and diluted 300-fold with 21H.

Test subject: Healthy, normal person, with the test being carried out immediately after the blood sample was taken.

Measuring conditions: according to the example, heating temperature 48ºC, heating time 2 min 40 sec, measuring time 4 min and n = 5.

As explained in detail above, the present invention relates to an improved method for automatically measuring glycated hemoglobin based on the principle of high pressure liquid chromatography, which comprises the following steps:

Preparing a series of blood samples taken from each patient as whole blood or blood cell layer in a sample holding device, collecting the samples immediately before measurement (10 sec - several min), mixing while diluting with a hemolizer/washing liquid containing a reagent for removing labile HbA1c, and injecting the liquid into a column of a high pressure liquid chromatograph at a specific time after the start of mixing. If necessary, heating of dilution until injection into the column.

The invention has the following advantages:

1. Compared with the traditional method of using a long high quality column, a faster measurement can be performed and the same level of accuracy can be achieved.

2. Labor related to manual mixing of reagents is saved, allowing accurate measurement without errors caused by manual dilution.

3. No pretreatment is required as whole blood or a blood cell layer in a centrifuged state is used.

4. Since the sample is not diluted by the hemolyzing reagent in advance, no changes occur in the sample until measurement, and stable HbA1c can be accurately measured by preventing the decomposition or change of glycated hemoglobin other than labile HbA1c.

5. Since the measured value is not influenced by the unstable HbA1c, there is no time restriction regarding the blood sampling, so that the patient is not burdened.

6. Since a sample is hemolyzed immediately before measurement and is always injected into the column at a constant time, consistent measurements can be performed.

In particular, the equilibrium state is not affected. Even if the samples are not used continuously, the patterns of the chromatogram are stable.

The sample injection valve heats the sample liquid and reagent to a specific temperature for a specific period of time. Since only the specimen is heated, no energy is wasted and there is no risk of injecting an unheated part into the column, resulting in precise control. The valve can be easily manufactured by improving a conventional sample injection valve. Since another heating device is not required, space is saved.

Claims (5)

1. Automatic measuring method for glycated hemoglobin based on the principle of high-pressure liquid chromatography, characterized by the following steps: Providing a series of blood samples (14) collected in sample vessels (6) as whole blood or as a blood cell layer, introducing the sample vessels (6) into a sampling part (2) to dilute the blood sample sucked from the sample sampling nozzle (9) by mixing it with a hemolyzing liquid (13) containing a reagent for removing labile HbA1c, supplying a portion of the mixture to a sample loop (18a) of a sample injection valve (18) kept at 30-65ºC, injecting it into a column (19) at a specific time after the start of mixing, and measuring the hemoglobin content in the state in which glycohemoglobin is not degraded and labile HbA1c is completely degraded. 2. Automatic measuring method for glycated haemoglobin according to claim 1, in which, in addition to the sample vessels (6) provided, a blood sample is taken from a sample vessel (6) which is located on a measuring opening (8) for urgent measurements. 3. Automatic measuring method for glycated hemoglobin according to claim 1, in which, in the case of a mixing start at the wrong time calculated back from the next injection time, a blank measurement without injection is carried out in order to inject the mixture into the column (19) at a specific time after the start of mixing of the blood sample with the hemolyzing liquid. 4. An automatic measuring method for glycated hemoglobin according to claim 1, wherein the hemolyzing liquid containing the reagent for removing labile HbA1c is used as a washing liquid which washes each flow line including the sample loop (18h) and a sample nozzle part. 5. Method according to claims 1 to 4, in which the sample loop part is surrounded by a loop heater (18b) and the whole is provided with an insulating cover (18c).